C端带His标签重组ScFv真核表达载体的构建及鉴定  

Construction and identification of ScFv eukaryotic expression vectors with His-tag at C terminal

作　　者：赖德华[1] 沈粤春[2] 赵超尘[1] 吴阳[1] 王晓明[1] 李君[1] 

机构地区：[1]广州医科大学附属第一医院肝胆外科,广东广州510120 [2]广州医科大学附属第一医院心内科,广东广州510120

出　　处：《广州医学院学报》2013年第5期13-16,共4页Academic Journal of Guangzhou Medical College

基　　金：广州市卫生局项目(20121A011140);广州市科技计划项目(2009J1-C371-1)

摘　　要：目的:构建表达抗p185且含His标签的ScFv的真核表达载体。方法:通过PCR自pcDNAH520C9ScFv-hIL-2质粒中获得H520C9ScFv片段,将此片段插入真核表达质粒pcDNA3.1(+)中;设计引物在ScFv片段C端增加His标签和凝血酶识别序列,再与质粒pcDNA3.1(+)连接,将所得的融合基因进行限制性内切酶EcoR I、HindⅢ双酶切及琼脂糖凝胶电泳鉴定,并测定目的序列。结果:成功将H5200C9ScFv片段、His标签和凝血酶识别序列插入真核表达质粒pcDNA3.1(+)中。结论:成功构建了真核表达载体pcDNA3.1-His-ScFv,为下一步在真核细胞中表达和纯化抗p185单抗奠定基础。Objective：To construct the ScFv eukaryotic expression vector with His-tag at C-terminal which possessed anti-p185 properties. Methods： The H520CgsFv fragment was derived from the pcDNA-H520CgScFv- hIL-2 plasmid by polymerase chain reaction and inserted into the eukaryotic expression plasmid pcDNA3.1 （ ＋ ）. The primers were designed to allow for addition of the His label and the thrombin-recognizing sequence to the C- terminal of ScFv, which entailed insertion into the eukaryotic expression vector pcDNA3.1 （ ＋ ）. The fusion gene was subject to EcoR I and Hind Ⅲ restriction enzyme digestion and agarose gel electrophoresis for sequencing of the targeted fragment. Results： The H520CgsFv fragment, His label and the thromhin-recognizing sequence have been successfully inserted into the eukaryotic expression vector pcDNA3.1 （ ＋ ）. Conclusion： The successful construction of the eukaryotic expression vector peDNA3.1-His-ScFv provides a solid basis for further research on expression and purification of anti-p185 monoclonal antibodies in eukaryotie cells.

关 键 词：载体构建 His标签 融合基因 乳腺肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]